The molecular chaperones, Hsp70 and Hsp90 bind to unfolded proteins (e.g. clients) and recruit either pro-folding or pro-degradation co-chaperones, generating a series of distinct multi- protein complexes that either fold or degrade the bound protein. For some important clients, such as microtubule-binding protein tau (MAPT/tau), a key step in the choice to degrade seems to be the dynamic recruitment of the E3 ubiquitin conjugating ligase CHIP, which couples both Hsp70 and Hsp90 to the ubiquitin-proteasome system (UPS). However, little is known about the molecular events that govern CHIP assembly into the complex and we do not yet understand why some disease-associated proteins, such as hyper-phosphorylated tau, evade this process. Progress towards understanding how clients are loaded into the pro-degradation complex has been hindered by a lack of structural information, the dynamic nature of the protein-protein interactions and the difficulties of linking in vitro findings with cellular functions. We hypothesize that a rigorous ad comprehensive understanding of protein triage will emerge from a combination of chemical crosslinking-mass spectrometry (CXL-MS), cryo-electron microscopy (cryo-EM) and new chemical probes that trigger an acute switch to the pro- degradation complex. Guided by strong preliminary results, we will: (SA1) generate a complete CXL-MS signature of the Hsp70-CHIP-tau and Hsp90-CHIP-tau complexes and map their protein-protein contacts in vitro and in intact neuronal cells, (SA2) use these signatures, chemical probes and mass spectrometry to understand how the complexes form and how they recruit CHIP and other effectors of the UPS during active triage in the cytosol and (SA3) elucidate the macromolecular architecture of the chaperone complexes to, for the first time, link cellular observations with careful measurements of protein-protein contacts in vitro. This work is significant because it will reveal changes in th composition of macromolecular complexes that drive active protein triage and it is innovative because it links powerful in vitro approaches with new chemical probes and CXL-MS to address one of the key questions in cellular protein homeostasis. Moreover, the proposed work brings together a continuum of research expertise in structural, chemical and cellular approaches to the study of dynamic protein-protein interactions.